Alpha 5 beta 1 and its ability to regulate the cell survival pathway

ABSTRACT

The present invention provides for identification of agents that induce growth arrest and survival of cancer cells, which remain dormant in bone marrow, thus preventing their eradication through use of standard chemotherapy or radiation therapy. Basic fibroblast growth factor (FGF-2), a mammary differentiation factor abundant in the bone marrow stroma, induces growth arrest of relatively differentiated breast cancer cells and restricts their survival to fibronectin by upregulating integrin α5β1. Most of the FGF-2-arrested cells fail to establish optimal ligation to fibronectin and undergo cell death. Cells that do attach to fibronectin, another major constituent of the bone marrow microenvironment, stay alive and growth-arrested for many weeks. Using function-blocking antibodies and peptides, a specific contribution of α5β1-fibronectin interaction in maintaining survival of growth-arrested cells was demonstrated. The present invention thus allows for methods, agents and pharmaceutical compositions that can be used to potentiate the activity of chemotherapy or radiation therapy.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a Divisional of copending non-provisionalapplication Ser. No. 10/521,841, which has a filing date under 35 U.S.C.§371(c), of Jul. 27, 2005, and is a National Stage filing of PCTApplication No. PCT/US2003/021954, filed Jul. 16, 2003, and which, inturn, claims priority from provisional application Ser. No. 60/396,482,filed on Jul. 16, 2002. Applicant claims the benefit of 35 U.S.C. §120as to said non-provisional application and said PCT application, and 35U.S.C. §119 as to said provisional application, and the disclosures ofall of said applications are hereby incorporated by reference herein intheir entireties.

GOVERNMENT RIGHTS CLAUSE

The research leading to the present invention was supported, at least inpart, by U.S. Army Grant No. DAMD17-01-C-0343. Accordingly, theGovernment may have certain rights in the invention.

FIELD OF THE INVENTION

The invention relates generally to the field of oncology, cancermetastasis and cellular proliferation. In particular, this inventionrelates to the identification of methods of interrupting certainelements of the cell survival pathway, which then allows for enhancedefficacy of traditional modes of cancer therapy, including chemotherapyand radiation therapy. More particularly, the invention relates to theuse of kinase or transcription inhibitors for pre-treatment to sensitizefor, or concurrent treatment to potentiate chemotherapy or radiationtherapy for treatment of cancers or hyperproliferative disorders. Theinvention also provides for the use of kinase or transcriptioninhibitors to downregulate expression of the alpha 5 beta 1 integrinand/or phosphorylation of Akt to treat cancer or hyperproliferativedisorders. Blocking antibodies specific for the alpha 5 beta 1 integrinare also envisioned for use in either pretreatment to sensitize for, orto be used concurrently with chemotherapy or radiation therapy fortreatment of cancer or hyperproliferative disorders. Methods oftreatment of cancer or hyperproliferative disorders using fibronectinbinding blocking peptides to sensitize for or potentiate chemotherapy orradiation therapy are also envisioned by the present invention.Furthermore, the invention relates to methods of use of retinoids todecrease the expression or phosphorylation of Akt and treatment ofcancer or hyperproliferative disorders. The instant invention alsoprovides for pharmaceutical compositions comprising, and methods ofusing the agents of the present invention for treatment of cancer orhyperproliferative disorders. Screening methods for identification ofnovel agents for use in treating cancer or hyperproliferative disordersin accordance with the present invention is also disclosed.

BACKGROUND OF THE INVENTION

Breast cancer cells metastasize to the bone marrow early in the courseof the disease (Braun, S., et al. (2000) The New England J. Med. 342,525-533). Most metastatic cells die upon reaching the marrowmicroenvironment, but some well-differentiated cells that survive canremain dormant, or growth arrested without loss of viability, for years(Boyce, B. F., et al. (1999) Endocrine-Related Cancer 6, 333-347; Chang,J., et al. (1999) J. Clinical Oncology 17, 3058-3063). They remainprotected from death and, in fact, survive multiple rounds of adjuvantchemotherapy administered specifically to eradicate them (Braun, S., etal. (2000) J. Clin. Onc. 18, 80-86). The factors and the mechanisms thatinduce dormancy, that is, growth arrest coupled with long-term survival,of occult breast cancer cells in bone marrow microenvironment and whichprotect the cells from chemotherapy remain largely unknown. However, avariety of growth factors and ligands of cellular integrins in themarrow microenvironment may influence the fate of the metastatic cell.These factors have well-established effects on cell behavior, includingprotection of hematopoietic stem cells (Ploemacher, R. E. (1997)Baillieres Clinical Haematology 10, 429-444; Knaan-Shanzer, S., et al.,(1999) Experimental Hematology 27, 1440-1450).

Bone marrow stroma is a rich source of growth factors such as epidermalgrowth factor (EGF), insulin-like growth factor (IGF-1) and basicfibroblast growth factor (FGF-2). FGF-2, a factor implicated in mammaryductal differentiation, induces growth arrest in a variety of relativelydifferentiated breast cancer cells.

However, there is a further need for identification of the factorsresponsible for growth arrest and long-term survival of occult cancercells, as well as a better understanding of the mechanisms involved.Upon identification of the factors involved, novel therapeutics may bedeveloped which could be used as stand-alone therapies or may be used asadjunct therapy with other standard forms of therapy to treat cancer orhyperproliferative disorders, such as chemotherapy or radiation therapy.It is with respect to this unmet need that the current invention isdirected.

Other advantages of the present invention will become apparent from theensuing detailed description taken in conjunction with the followingillustrative drawings.

SUMMARY OF THE INVENTION

It is known that malignant cells from breast cancer micrometastases aswell as other hyperproliferative disorders in bone marrow remain dormantwithout loss of viability for prolonged periods of time. It is in thisgrowth arrested state that the cells are resistant to standard forms oftherapy including chemotherapy or radiation therapy. The factors thatinduce this dormancy are unknown at this time. It is thus an object ofthe present invention to identify the factors responsible for thisdormancy, and to utilize these factors for identification, use of, andscreening for new therapeutic regimens for treatment of cancer and otherhyperproliferative disorders.

A first aspect of the invention provides for the identification and useof kinase or transcription inhibitors as pre-treatment or concurrenttreatment, to sensitize for or potentiate chemotherapy in the treatmentof cancer or hyperproliferative disorders. In a preferred embodiment,the agents identified by the present invention are inhibitors of MAPkinase, Rho kinase, PI3 kinase and/or PKC kinase.

In another preferred embodiment, the kinase or transcription inhibitorsare used to treat metastatic cancers and/or hyperproliferativedisorders. In another preferred embodiment, the kinase or transcriptioninhibitors are used to treat breast cancer. In yet another preferredembodiment, the kinase or transcription inhibitors are used to treatmetastatic breast cancer.

In a second aspect of the invention, the kinase or transcriptioninhibitors are used to downregulate expression of the alpha 5 beta 1integrin.

In a third aspect of the invention, the kinase or transcriptioninhibitor decreases expression and/or phosphorylation of Akt and isutilized for treatment of cancer or hyperproliferative disorders.

A fourth aspect of the invention provides for the use of antibodies tointegrin alpha 5 beta 1 as pretreatment or concurrent treatment tosensitize for, or potentiate chemotherapy or radiation therapy in thetreatment of cancer or hyperproliferative disorders.

In a preferred embodiment, the antibodies are used to treat metastaticcancers or other hyperproliferative disorders. In another preferredembodiment, the antibodies are used to treat breast cancer. In yetanother preferred embodiment, the antibodies are used to treatmetastatic breast cancer. The antibodies may be polyclonal ormonoclonal. They may be single chain antibodies. They may be chimericantibodies. They may be Fab fragments or soluble components thereof.They may be human or humanized. They may be produced in other animals,including but not limited to horses, goats, sheep, mice, rats, rabbitsand guinea pigs.

A fifth aspect of the invention provides for the use of fibronectinbinding blocking peptides as pretreatment or concurrent treatment, tosensitize for or potentiate chemotherapy or radiation therapy in thetreatment of cancer or hyperproliferative disorders.

In a preferred embodiment, the fibronectin binding blocking peptides areused to treat breast cancer. In yet another preferred embodiment, thefibronectin binding blocking peptides are used to treat metastaticbreast cancer.

A sixth aspect of the invention provides for the use of retinoids todecrease expression or phosphorylation of Akt and treatment of cancersor hyperproliferative disorders.

In a preferred embodiment, the retinoids are used to treat metastaticcancers or other hyperproliferative disorders. In another preferredembodiment, the retinoids are used to treat breast cancer. In yetanother preferred embodiment, the retinoids are used to treat metastaticbreast cancer.

A seventh aspect of the invention provides for a method of inhibitingcellular proliferation in a mammal suffering from a disease or adisorder characterized by cellular proliferation, the method comprisingadministering an effective amount of a kinase or transcription inhibitorprior to, or concurrent with chemotherapy or radiation therapy. In apreferred embodiment, the kinase inhibitor is selected from the groupconsisting of LY294002, UO 126, AG82, Y27632, SB203580, PD169316,PD98059, RO318220, or C3 transferase inhibitor.

In another preferred embodiment, the disease or disorder characterizedby cellular proliferation is cancer or a hyperproliferative disorder. Inanother preferred embodiment, the cancer is a metastatic cancer. Inanother preferred embodiment, the cancer is breast cancer. In yetanother preferred embodiment, the breast cancer has metastasized.

In a yet further preferred embodiment, the kinase or transcriptioninhibitor downregulates expression of the alpha 5 beta 1 integrin orphosphorylation of Akt to sensitize for or potentiate chemotherapy orradiation therapy in mammals in need thereof.

An eighth aspect of the invention provides for a method for disruptingsurvival signaling from the microenvironment in cancer cells, whereinsaid disrupting results in sensitizing cells to chemotherapy, biologicaltherapies or radiation therapy of cancer micrometastases andhyperproliferative disorders in a mammal. In a preferred embodiment, theintegrin is alpha 5 beta 1 and the extracellular matrix protein isfibronectin. In another preferred embodiment, the cancer is breastcancer or prostate cancer. In yet another preferred embodiment, themethod comprises administration of an antibody specific for an integrinor a blocking peptide or modified peptide that disrupts interaction ofthe integrin with the extracellular matrix. In a yet further preferredembodiment, the method comprising administration of all trans retinoicacid or a retinoic acid derivative. A yet further embodiment comprisesdecreasing expression of cell surface integrins with a transcriptioninhibitor, or blocking survival signaling initiated by ligation ofintegrins by microenvironment proteins. A most preferred embodimentprovides for treatment with an inhibitor of a kinase, said kinaseselected from the group consisting of MAP kinase, Rho kinase, PI3 kinaseand PKC kinase. The most preferred inhibitors are selected from thegroup consisting of LY294002, UO 126, AG82, Y27632, SB203580, PD169316,PD98059, RO318220, and a 3 transferase inhibitor.

A ninth aspect of the invention provides for a method for treatinghyperproliferative disorders in a mammal, comprising administration ofan agent capable of blocking the binding of integrins with theextracellular matrix. In a preferred embodiment the integrins comprisesalpha 5 beta 1 and the matrix is fibronectin.

A tenth aspect of the invention provides for the use of an agent for thepreparation of a composition for treatment of hyperproliferativedisorders, said agent capable of downregulation of the expression of thealpha 5 beta 1 integrin and its binding to the extracellular matrix.

An eleventh aspect of the invention provides for pharmaceuticalcompositions comprising the kinase or transcription inhibitor and apharmaceutically acceptable carrier, or an antibody, blocking peptide ormodified peptide and a pharmaceutically acceptable carrier.

Other objects and advantages will become apparent from a review of theensuing detailed description and attendant claims. All references citedin the present application are incorporated herein in their entirety.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Breast cancer cells that metastasize to the bone marrow arearrested by deposits of FGF-2 in the bone marrow stroma. FGF-2 inducesoverexpression of integrins α5 and β1 and leads to massive cell deaththrough unligated or inappropriately ligated integrins by a processtermed integrin-mediated death (IMD). Adherence and appropriate ligationof α5β1 on the surviving cells by fibronectin in the stroma interruptsIMD and initiates survival signaling through PI3K. This results indormancy of the non-cycling cells and protection from cell death inducedby cytotoxins. Disruption of the integrin-fibronectin interaction woulddiscontinue survival signaling and initiate IMD. This would render themetastatic cells sensitive to chemotherapy.

FIG. 2. Survival effects of fibronectin on FGF-2-inhibited breast cancercells

MCF-7 cells were incubated at a concentration of 1000 cells/well on 24well plates coated with A. collagen I or B. fibronectin or laminin I forvariable periods from 3 to 9 days, stained with crystal violet andphotographed. A. Colony cultures in collagen I-coated dishesdemonstrating increased colony formation with time in the two controlrows and with 10 ng/ml EGF-treatment and almost complete abolition ofcolony formation by FGF-2 10 ng/ml. Experiments were done at least twicewith similar results. B. Similar cellular obliteration was observed onlaminin I-coated plates, but incubation on fibronectin yielded survivalof a small number of nonproliferating cells. Shown are typical 5 daycolonies in control wells and impaired colony formation in wellscontaining FGF-2 10 ng/ml.

FIG. 3. FGF-2 inhibits the clonogenicity of MCF-7 cells but has noeffect on MDA-MB-231 cell

One thousand MCF-7 or MDA MB-231 cells per well were incubated in 24well tissue culture plates with and without the presence of 10 ng/mlbasic fibroblast growth factor (FGF-2) or epidermal growth factor (EFG).Plates were stained with crystal violet after a 5 day incubation andclones consisting of A. 29±2 cells and B. 8±2 cells were counted. C. Tenthousand T-47D cells per well were incubated in 24 well tissue cultureplates coated with fibronectin with and without the presence of 10 ng/mlbasic fibroblast growth factor (FGF-2) or epidermal growth factor (EFG).Plates were stained with crystal violet after 3 days and clonesconsisting of 8 or more cells were counted.

FIG. 4. Cloning efficiency of MCF-7 cells in the presence of FGF-2

Five thousand MCF-7 cells were incubated per well in duplicate on 6 welltissue culture dishes with various substrata with FGF-2 10 ng/ml.Colonies of 8 cells or greater were counted after staining the plateswith crystal violet after 5, 10 and 15 day incubations. Incubation onfibronectin continued to preserve the clonogenicity of these cell linesfor up to the 15 days assayed.

FIG. 5. Nonrad GEArray Q series gene chip microarray analysis of MCF-7cells incubated with and without FGF-2 for 5 days on tissue culturedishes coated with fibronectin

Gene chip microarray analysis of MCF-7 cells incubated for 5 days ontissue culture dishes coated with fibronectin 20 μg with and without thepresence of FGF-2 10 ng/ml. Approximately one third as many cellsremained in the FGF-2-treated population as in the control cells. A.Nonrad GEArray Q series Human Extracellular Matrix and Adhesion Proteinchip (Super Array, Bethesda, Md.). Arrows point to integrin α5 (solidline) and α6 (dotted line) mRNA's that are elevated in the survivingpopulation. Boxes are drawn around the control gene cDNAs on the twochips consisting of GAPDH, Cyclophyllin A, ribosomal L23 and β actin aspositive controls and PUC18 plasmid DNA and blanks as negative controls.B. Nonrad GEArray Q series Human Pathway Finder chip (Super Array,Bethesda, Md.). Arrow points to the p16^(INK4) gene whose expression isdownregulated by FGF-2 treatment on fibronectin. Numbers on right ofchips indicate the numbering of the rightmost member cDNA of each row.

Changes in gene expression due to FGF-2 treatment on afibronectin-coated plate for five days were observed in the followinggenes on the two chips noted in Table 1 and Table 2.

FIG. 6. FGF-2 regulates expression of integrins.

A. Gene chip analysis of integrin α5 and β1 mRNA expression in MCF-7cells incubated±FGF-2 for 3 or 5 days on fibronectin-coated plates.Densitometer quantitations normalized against GAPDH and actin mRNAstandards are shown. B. Western blots of integrin α5 from cellsincubated±FGF-2 for 3 days on tissue culture- or fibronectin-coateddishes. C. Indirect immunofluorescence of integrin α5 in T-47D cells oncover slips±FGF-2 10 ng/ml for 24 hours. D. Western blots of integrinsα2, α3, α4, α6, β1, β3 and β4 in MCF-7 and T-47D cells incubated±FGF-2for 3 days. Nonspecific bands were used as loading controls.

FIG. 7. Integrin α5-dependent clonogenic survival of MCF-7 cells onfibronectin

Five thousand MCF-7 cells were incubated per well in quadruplicate on5-well tissue culture dishes with and without 10 ng/ml FGF-2, in thepresence or absence of 2 μg neutralizing mouse monoclonal antibody tointegrin α5 or integrin α3 (Chemicon, Inc, Temecula, Calif.). Cells werecultured for 5 days, stained with crystal violet and clones with 8±2cells were counted.

FIG. 8. Fibronectin-specific blocking peptides selectively inhibitclonogenicity on fibronectin.

10³ MCF-7 (and T-47D, not shown) cells were incubated±fibronectin with10 ng/ml FGF-2. Fibronectin-blocking peptide GRGDSP 1 ng/ml (AmericanPeptide Co., Inc, Sunnyvale, Calif.) was added after 3 days and 4, 8 and12 cell colonies were counted 6 days later. Blocking peptide onlyinhibited colonies on fibronectin, and not on plastic.

FIG. 9. Ligation of Integrin α5β1 provides specific protection from celldeath in well-differentiated breast cancer cells

A. MCF-7 cells (and T-47D cells, not shown) were incubated with FGF-2 onvariably coated plates. Blocking peptides were added after 3 days.Colonies with ≦10 cells were stained with crystal violet at 6 days andcounted. B. T-47D cells were incubated on fibronectin-coated plates withFGF-2 and blocking peptides were added after 3 days. Cells were probed24 hours later with anti-integrin α5 antibody and Texas Red-taggedsecondary antibody and assayed by TUNEL-FITC.

FIG. 10. Induction of sustained Akt phosphorylation by FGF-2 onfibronectin

Western blots of lysates from MCF-7, T-47D and MDA-MB-231 cellsincubated on fibronectin-coated plates with FGF-2 for up to 5 days werestained with antibody to phospho-Akt or total Akt. Blots show sustainedphosphorylation of Akt by FGF-2 in MCF-7 and T-47D cells but no effecton constitutive Akt phosphorylation in MDA-MB-231 cells. No effect wasnoted on total Akt levels. Stained membrane was used as a loadingcontrol.

FIG. 11. All-trans retinoic acid dampens EGF-mediated AKTphosphorylation

MCF-7 cells were treated with EGF 100 ng/ml for 10 min followed by ATRA10⁻⁷ M or control media 2 h later for an additional 24 h. Western blotsof lysates were stained with anti-phospho-Akt ab.

FIG. 12. Effects of EGF and FGF-2 on the clonogenic potential of welland poorly-differentiated breast cancer cells in tissue culture

MCF-7 and T-47D (1,000 cells/well) and MDA MB-231 (200 cells per well)were incubated in 24 well plates±10 ng/ml EFG or FGF-2 for 6 days,stained with crystal violet and clones with ≧29 actively growing cells(▪) or with ≦10 well spread, growth arrested cells (

) were counted.

FIG. 13. Adhesion of breast cancer cells to stromal proteins

Both MCF-7 and T-47D cells were cultured±FGF-2 on tissue culture (A) oron fibronectin-coated plates (B), detached with Cell DissociationSolution, washed with PBS and counted. Cells were incubated with 2 μg/mlblocking monoclonal antibodies to the integrins or mouse IgG for 30minutes at 37° C. and 50,000 cells were incubated in 24 wellvariably-coated tissue culture plates for 45 minutes at 37° C. Attachedcells were stained with crystal violet and the A₆₀₀ of the extracted dyewas measured, as described. Results were similar for both cells. Shownare data for T-47D (A) and MCF-7 cells (B). Antibody to integrinαblocked adhesion to fibronectin in FGF-2 treated cells by 75% but onlyinhibited untreated cell adhesion by a third. Blocking antibody to α2decreased adhesion to collagen and laminin in both FGF-2 treated anduntreated cells equally. While adhesion to collagen surpassed adhesionto fibronectin, it did not support dormant clone survival. Theseadhesion controls demonstrated that the data are consistent with aspecific survival effect derived from ligation to fibronectin in dormantcells and not merely an effect due to nonspecific adhesion.

FIG. 14. Stroma restricts growth of well-differentiated T-47D breastcancer cells.

A. Confluent stromal cultures in 24 well plates seeded with 500 T-47D orMDA-MB-231 cells/well were cultured for 6 days. B. Cytokeratin 19immunofluorescence (red) staining of MCF-7 cells on stromal co-culture(blue background) demonstrating a primarily single cell status of MCF-7cells after 6 days. C. Western blots of stromal cell lysates (100 μg)with recombinant FGF-2 and lysates from T-47D cells transfected with avector expressing 18, 22, 22.5 and 24 kD FGF-2 isoforms. D. MCF-7 cellswere seeded on stromal monolayers on 24 well plates (1,000 cells/well).Blocking peptides were added after 3 days. At 6 days, plates werestained with anti-cytokeratin 19 antibody and horseradishperoxidase-tagged secondary antibody, developed and colonies of ≦10cells counted.

FIG. 15. Akt-inhibitor reduces fibronectin-promoted survival of dormantbreast cancer cell clones.

MCF-7 and T-47D cells were incubated with FGF-2 on fibronectin for threedays, media was changes and supplemented with variable concentrations ofinhibitor and fresh FGF-2 and incubated for an additional three days.Colonies≦10 cells were counted after crystal violet staining. Data isplotted as percent change from colony numbers on tissue culture coatedplastic dishes.

FIG. 16. The phosphatidyl inositol 3-kinase (PI3 kinase) inhibitorLY294002 reduces fibronectin-promoted survival of dormant breast cancercell clones.

MCF-7 and T-47D cells were incubated with FGF-2 on fibronectin for threedays, media was changed and supplemented with variable concentrations ofinhibitor and fresh FGF-2 and incubated for an additional three days.Colonies≦10 cells were counted after crystal violet staining.

FIG. 17. Inhibition of dormant clone survival by kinase inhibitors.

A. MCF-7 cells (and T-47D cells, not shown) and B. T-47D cells wereincubated with FGF-2 on fibronectin for three days, media was changedand supplemented with variable concentrations of a variety of kinaseinhibitors and a small GTPase inhibitor and fresh FGF-2, and incubatedfor an additional three days. Control cells were incubated in 10 μM DMSOas control for the solvent used with the inhibitors. Colonies≦10 cellswere counted after crystal violet staining. Data are plotted as percentchange from colony numbers on tissue culture coated plastic dishesdemonstrating significant inhibition of dormant clones by abrogating anumber of signaling pathways. C. The inhibitors used were:

Inhibitor Target ED50 UO 126 MEK 1 72 nM MEK 2 58 nM AG82 FAK 7 μMY27632 Rho kinase 140 nM SB203580 p38 600 nM PD169316 p38 89 nM PD98059MEK 2 μM RO318220 Protein kinase C 10 nM Protein kinase A 900 nM C3transferase RhoA 2-5 μg/ml inhibitor

DETAILED DESCRIPTION

Before the present methods and treatment methodology are described, itis to be understood that this invention is not limited to particularmethods, and experimental conditions described, as such methods andconditions may vary. It is also to be understood that the terminologyused herein is for purposes of describing particular embodiments only,and is not intended to be limiting, since the scope of the presentinvention will be limited only in the appended claims.

As used in this specification and the appended claims, the singularforms “a”, “an”, and “the” include plural references unless the contextclearly dictates otherwise. Thus, for example, references to “themethod” includes one or more methods, and/or steps of the type describedherein and/or which will become apparent to those persons skilled in theart upon reading this disclosure and so forth.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the invention, the preferred methods andmaterials are now described. All publications mentioned herein areincorporated herein by reference.

Definitions

The terms used herein have the meanings recognized and known to those ofskill in the art, however, for convenience and completeness, particularterms and their meanings are set forth below.

“Agent” refers to all materials that may be used to preparepharmaceutical and diagnostic compositions, or that may be compounds,nucleic acids, polypeptides, fragments, isoforms, variants, or othermaterials that may be used independently for such purposes, all inaccordance with the present invention.

The term “antibody” as used herein includes intact molecules as well asfragments thereof, such as Fab and F(ab′)₂, which are capable of bindingthe epitopic determinant. Antibodies that bind the proteins of thepresent invention can be prepared using intact polypeptides or fragmentscontaining small peptides of interest as the immunizing antigen attachedto a carrier molecule. Commonly used carriers that are chemicallycoupled to peptides include bovine serum albumin and thyroglobulin. Thecoupled peptide is then used to immunize the animal (e.g, a mouse, rator rabbit). The antibody may be a “chimeric antibody”, which refers to amolecule in which different portions are derived from different animalspecies, such as those having a human immunoglobulin constant region anda variable region derived from a murine mAb. (See, e.g., Cabilly et al.,U.S. Pat. No. 4,816,567; and Boss et al., U.S. Pat. No. 4,816,397.). Theantibody may be a human or a humanized antibody. The antibody may be asingle chain antibody. The antibody may be prepared in mice, rats,rabbits, goats, sheep, swine, dogs, cats, or horses.

“Analog” as used herein, refers to a chemical compound, a nucleotide, aprotein, or a polypeptide that possesses similar or identical activityor function(s) as the chemical compounds, nucleotides, proteins orpolypeptides having the desired activity and therapeutic effect of thepresent invention (eg. to inhibit cellular proliferation and tosensitize for, or potentiate chemotherapy or radiation therapy fortreatment of mammals having cancer or hyperproliferative disorders), butneed not necessarily comprise a sequence that is similar or identical tothe sequence of the preferred embodiment, or possess a structure that issimilar or identical to the agents of the present invention. As usedherein, a nucleic acid or nucleotide sequence, or an amino acid sequenceof a protein or polypeptide is “similar” to that of a nucleic acid,nucleotide or protein or polypeptide having the desired activity if itsatisfies at least one of the following criteria: (a) the nucleic acid,nucleotide, protein or polypeptide has a sequence that is at least 30%(more preferably, at least 35%, at least 40%, at least 45%, at least50%, at least 55%, at least 60%, at least 65%, at least 70%, at least75%, at least 80%, at least 85%, at least 90%, at least 95% or at least99%) identical to the nucleic acid, nucleotide, protein or polypeptidesequences having the desired activity as described herein (b) thepolypeptide is encoded by a nucleotide sequence that hybridizes understringent conditions to a nucleotide sequence encoding at least 5 aminoacid residues (more preferably, at least 10 amino acid residues, atleast 15 amino acid residues, at least 20 amino acid residues, at least25 amino acid residues, at least 40 amino acid residues, at least 50amino acid residues, at least 60 amino residues, at least 70 amino acidresidues, at least 80 amino acid residues, at least 90 amino acidresidues, at least 100 amino acid residues, at least 125 amino acidresidues, or at least 150 amino acid residues) of the AAPI; or (c) thepolypeptide is encoded by a nucleotide sequence that is at least 30%(more preferably, at least 35%, at least 40%, at least 45%, at least50%, at least 55%, at least 60%, at least 65%, at least 70%, at least75%, at least 80%, at least 85%, at least 90%, at least 95% or at least99%) identical to the nucleotide sequence encoding the polypeptides ofthe present invention having the desired therapeutic effect. As usedherein, a polypeptide with “similar structure” to that of the preferredembodiments of the invention refers to a polypeptide that has a similarsecondary, tertiary or quarternary structure as that of the preferredembodiment. The structure of a polypeptide can determined by methodsknown to those skilled in the art, including but not limited to, X-raycrystallography, nuclear magnetic resonance, and crystallographicelectron microscopy.

“Derivative” refers to either a protein or polypeptide that comprises anamino acid sequence of a parent protein or polypeptide that has beenaltered by the introduction of amino acid residue substitutions,deletions or additions, or a nucleic acid or nucleotide that has beenmodified by either introduction of nucleotide substitutions ordeletions, additions or mutations. The derivative nucleic acid,nucleotide, protein or polypeptide possesses a similar or identicalfunction as the parent polypeptide. It may also refer to chemicallysynthesized organic molecules that are functionally equivalent to theactive parent compound, but may be structurally different. It may alsorefer to chemically similar compounds which have been chemically alteredto increase bioavailability, absorption, or to decrease toxicity.

“Fragment” refers to either a protein or polypeptide comprising an aminoacid sequence of at least 5 amino acid residues (preferably, at least 10amino acid residues, at least 15 amino acid residues, at least 20 aminoacid residues, at least 25 amino acid residues, at least 40 amino acidresidues, at least 50 amino acid residues, at least 60 amino residues,at least 70 amino acid residues, at least 80 amino acid residues, atleast 90 amino acid residues, at least 100 amino acid residues, at least125 amino acid residues, at least 150 amino acid residues, at least 175amino acid residues, at least 200 amino acid residues, or at least 250amino acid residues) of the amino acid sequence of a parent protein orpolypeptide, or a nucleic acid comprising a nucleotide sequence of atleast 10 base pairs (preferably at least 20 base pairs, at least 30 basepairs, at least 40 base pairs, at least 50 base pairs, at least 50 basepairs, at least 100 base pairs, at least 200 base pairs) of thenucleotide sequence of the parent nucleic acid. Any given fragment mayor may not possess a functional activity of the parent nucleic acid orprotein or polypeptide.

A “therapeutically effective amount” is an amount sufficient to decreaseor prevent the symptoms associated with the cancer or hyperproliferativedisorders or other related conditions contemplated for therapy with thecompositions of the present invention.

“Treatment” refers to therapy, prevention and prophylaxis andparticularly refers to the administration of medicine or the performanceof medical procedures with respect to a patient, for either prophylaxis(prevention) or to cure or reduce the extent of or likelihood ofoccurrence of the infirmity or malady or condition or event in theinstance where the patient is afflicted.

“Combination therapy” refers to the use of the agents of the presentinvention with other active agents or treatment modalities, in themanner of the present invention for treatment of cancers orhyperproliferative disorders. These other agents or treatments mayinclude drugs such as other anti-cancer drugs such as those that arestandardly used to treat various cancers, radiation therapy, anti-viraldrugs, corticosteroids, non-steroidal anti-inflammatory compounds, otheragents useful in treating or alleviating pain, growth factors,cytokines, or colony stimulating factors. The combined use of the agentsof the present invention with these other therapies or treatmentmodalities may be concurrent, or the two treatments may be divided upsuch that the agent of the present invention may be given prior to orafter the other therapy or treatment modality.

“Local administration” means direct administration by a non-systemicroute at or in the vicinity of the site of an affliction, disorder, orperceived pain.

“Slow release formulation” refers to a formulation designed to release atherapeutically effective amount of a drug or other active agent such asa polypeptide or a synthetic compound over an extended period of time,with the result being a reduction in the number of treatments necessaryto achieve the desired therapeutic effect. In the matter of the presentinvention, a slow release formulation would decrease the number oftreatments necessary to achieve the desired effect in terms ofinhibiting cellular proliferation and decreasing the tumor burden ormetastatic potential of a cancer or hyperproliferative disorder.

The term “clonogenic potential” refers to the ability of single cells todivide and grow into a cluster of cells. This is a characteristic ofmetastatic cancer cells in the body. In the lab, it is a reflection ofmany factors, including viability, health of the cell, injury, andability to divide on the support provided in the tissue culture dish orin suspension

“EGF” is epidermal growth factor; a protein that binds to cell surfacereceptors and initiates signals that tell the cell to divide, crawl andsurvive.

“IGF” is insulin-like growth factor; a protein that binds to theinsulin-like growth factor receptor that initiates signals that tell thecell to do perform a variety of function from cell division survival,depending on the cell type.

“FGF-2” is fibroblast growth factor 2, basic fibroblast growth factor; aprotein that binds to cell surface receptors that initiates a variety ofsignals that tell different cells to perform different functions. Inbreast cancer, it can act as a differentiation factor, inhibiting growthand motility.

The term “hyperproliferative disorders” refers to diseases that resultfrom the abnormal growth of cells. These can include cancers,pre-malignant states as well as inflammatory states such as rheumatoidarthritis or conditions such as psoriasis.

“Integrins” are intrinsic cell surface proteins. They mediate celladhesion by binding with components of the extra cellular matrix, suchas fibronectin. This adhesion process is closely tied to the cellsability to survive and reproduce. Many different integrins have beendiscovered and most have similar structural features eg. they areheterodimeric transmembrane proteins and contain an alpha subunit and abeta subunit. The major fibronectin receptor on most cells is the alpha5, beta 1 integrin, also referred to in the present application as α5β1.This integrin interacts with the RGD site of the fibronectin molecule.

A kinase is a protein that acts as an enzyme to transfer a phosphategroup onto another protein. A “kinase inhibitor” blocks the action ofsuch a protein

A “transcription inhibitor” is a chemical or biological that interfereswith the synthesis of messenger RNA from a DNA template.

“ATRA” refers to all-trans retinoic acid; a member of a family ofcompounds called retinoids that act by binding to nuclear receptorscalled retinoic acid receptors and retinoid X receptors that, when boundto their retinoid ligands, act as transcription factors. ATRA inhibitscell proliferation, induces cell death and potentiates chemotherapyagents in breast cancer cells.

As used herein, the term “modified peptide” may be used to refer to apeptide that is capable of binding to a protein and modulating itsactivity (e.g., a cell surface receptor). Modified peptides may possessfeatures that, for example, modulate (increase or decrease) binding,alter the half-life of the peptide, decrease renal clearance, or improveabsorption.

As used herein, the term “amino acid” and any reference to a specificamino acid is meant to include naturally occurring proteogenic aminoacids as well as non-naturally occurring amino acids such as amino acidanalogs. One of skill in the art would know that this definitionincludes, unless otherwise specifically indicated, naturally occurringproteogenic (D) or (L) amino acids, chemically modified amino acids,including amino acid analogs such as penicillamine(3-mercapto-D-valine), naturally occurring non-proteogenic amino acidssuch as norleucine and chemically synthesized compounds that haveproperties known in the art to be characteristic of an amino acid. Asused herein, the term “proteogenic” indicates that the amino acid can beincorporated into a protein in a cell through well-known metabolicpathways.

The choice of including an (L)- or a (D)-amino acid into a peptide ofthe present invention depends, in part, on the desired characteristicsof the peptide. For example, the incorporation of one or more (D)-aminoacids can confer increasing stability on the peptide in vitro or invivo. The incorporation of one or more (D)-amino acids also can increaseor decrease the binding activity of the peptide as determined, forexample, using the binding assays described herein, or other methodswell known in the art. In some cases it is desirable to design a peptidewhich retains activity for a short period of time, for example, whendesigning a peptide to administer to a subject. In these cases, theincorporation of one or more (L)-amino acids in the peptide can allowendogenous peptidases in the subject to digest the peptide in vivo,thereby limiting the subject's exposure to an active peptide.

As used herein, the term “amino acid equivalent” refers to compoundswhich depart from the structure of the naturally occurring amino acids,but which have substantially the structure of an amino acid, such thatthey can be substituted within a peptide which retains is biologicalactivity. Thus, for example, amino acid equivalents can include aminoacids having side chain modifications or substitutions, and also includerelated organic acids, amides or the like. The term “amino acid” isintended to include amino acid equivalents. The term “residues” refersboth to amino acids and amino acid equivalents.

As used herein, the term “peptide” is used in its broadest sense torefer to compounds containing amino acid equivalents or other non-aminogroups, while still retaining the desired functional activity of apeptide. Peptide equivalents can differ from conventional peptides bythe replacement of one or more amino acids with related organic acids(such as PABA), amino acids or the like or the substitution ormodification of side chains or functional groups.

It is to be understood that limited modifications can be made to apeptide without destroying its biological function. Thus, modificationof a peptides of the present invention that does not completely destroyits activity are within the definition of the compound claims as such.Modifications can include, for example, additions, deletions, orsubstitutions of amino acids residues, substitutions with compounds thatmimic amino acid structure or functions, as well as the addition ofchemical moieties such as amino or acetyl groups. The modifications canbe deliberate or accidental, and can be modifications of the compositionor the structure.

An exemplary cell surface receptor envisioned for targeting by a peptideor “modified peptide” of the invention is a member of the integrinreceptor family. In an embodiment of the invention, a “modified peptide”may be used to inhibit integrin receptor activity, including, withoutlimitation, the ability of integrin-expressing cells to bind toextracellular matrix proteins and surrounding cells. Modified peptidescapable of inhibiting integrin binding/activity have been described inU.S. Pat. Nos. 5,536,814; 5,627,263; 5,912,234; 5,922,676; 5,981,478;5,912,234; and 6,177,542, the entire contents of each of which is hereinincorporated in its entirety by reference.

Retinoids are a class of compounds consisting of four isoprenoid unitsjoined in a head-to-tail manner. All retinoids may be formally derivedfrom a monocyclic parent compound containing five carbon-carbon doublebonds and a functional group at the terminus of the acyclic portion.Derivatives of retinoids may be generated by means known to skilledartisans to render the retinoid derivative more therapeuticallyeffective. A retinoid derivative may be, for example, an aldehydederivative, a carboxylic acid derivative, a substituted derivative, ahydrogenated derivative, or it may be derivatized by functionalsubstitution of a basic hydrocarbon. Retinoid derivatives may, forexample, be generated that are more specifically targeted tohyperproliferative cells. As used herein, the term “retinoid derivative”may also be used to refer to a compound or agent having retinoidactivity, but which does not necessarily act through a retinoidreceptor.

As used herein, the term “biological therapy” refers to a therapeuticregimen designed to enhance a subject's or patient's response totreatment administered to reduce the number of cancer cells and/orsymptoms associated with cancer. In general, “biological therapy”involves the use of a variety of cytokines, including, but not limitedto, growth factors, interferons, colony stimulating factors, tumornecrosis factors, and interleukins.

As used herein, the term “sensitization” or “sensitizing” refers totreating a subject so as to render the subject or cells therein moresusceptible to the effects of a therapeutic regimen. A number ofsensitizing agents have been characterized that render cancer cells, forexample, more susceptible to therapeutic modalities designed toeradicate cancer from a subject. Such sensitizing agents have beenpreviously described in, for example, U.S. Pat. No. 5,436,337, theentire contents of which is incorporated herein by reference in itsentirety.

As used herein, the phrase “disrupting survival signaling from themicroenvironment” refers to a situation in which interactions betweenintegrins and their ligands are reduced or decreased. Such interactionsmay be physically blocked using antibodies or peptides; or may beprevented by decreasing the cell surface expression levels of integrinsvia transcriptional inhibition; or by blocking survival signalinginitiated by integrin receptor ligation by proteins in themicroenvironment.

General Description

The present invention relates to the novel finding that increasedexpression of the alpha-5 beta-1 integrin on metastasized breast cancercells in the bone marrow transmits a survival signal from matrixproteins in the bone marrow. Ligation of the integrin to fibronectininterrupts integrin-mediated cell death signaling and initiates the cellsurvival signaling that leads to dormancy, protection from chemotherapyand ultimately relapse in the breast cancer patient. The inventionprovides for a method to inhibit the expression of the integrin and tointerrupt specific elements of the survival pathway that will allowtraditional chemotherapy or radiation therapy to be utilized to kill theremaining cells in the bone marrow and avoid a relapse and ultimatelyresistance by the cells and the death of the patient suffering from ahyperproliferative disorder such as but not limited to breast cancer, orprostate cancer. The over expression of alpha-5 beta-1 is down regulatedthrough the use of kinase or transcription inhibitors such asdemonstrated in FIG. 1.

The schema of FIG. 1 demonstrates the fate of metastatic cells in thebone marrow and the effect of fibronectin ligation through its integrinreceptor alpha5 beta1 on maintaining survival and chemoresistance.Disruption of this interaction by decreasing synthesis of the integrinor disruption of its interactions with its ligand would allow the cellsto become sensitive to chemotherapy and undergo cell death.

In the present invention, evidence is provided which supports a paradigmin which FGF-2 initiates a more differentiated, dormant state inwell-differentiated micrometastatic breast cancer cells. Thisencompasses cell cycle arrest and changes in the integrin repertoire.Cells with improperly ligated integrins such as α5β1, upregulated byFGF-2 in fibroblasts and endothelial cells undergo cell death, likelydue to ligand incompatibility. Ligation of integrin α5β1 by fibronectin,a component of bone marrow stroma, which can initiate survival signaling(Matter, M. L, & Ruoslahti, E. (2001) J. Biol. Chem. 276, 27757-27763;Lee, J. W. & Juliano, R. L. (2000) Molecular Biology of the Cell 11,1973-1987), promotes survival of FGF-2-responsive cells.

In particular, the present invention is directed to methods fordisrupting survival signaling from the microenvironment in cancer cells,wherein said disrupting results in sensitizing cells to chemotherapy,biological therapies or radiation therapy of cancer micrometastases andhyperproliferative disorders in a mammal. The method comprises blockingthe interaction of an integrin with an extracellular matrix protein ofthe microenvironment. A preferred embodiment includes the alpha 5 beta 1integrin and the preferred extracellular matrix protein is fibronectin.The invention is directed to treating primary tumors, tumor metastasis,micrometastases and hyperproliferative disorders. A further preferredembodiment is treating breast cancer or prostate cancer.

A further preferred embodiment comprises administration of an antibodyspecific for an integrin or a blocking peptide or modified peptide thatdisrupts interaction of the integrin with the extracellular matrix. Ayet further preferred embodiment comprises administration of all transretinoic acid or a retinoic acid derivative. A yet further preferredembodiment comprises decreasing expression of cell surface integrinswith a transcription inhibitor. The method also comprises treatment withan inhibitor of a kinase, said kinase selected from the group consistingof MEP/MAP kinase, p38, RhoA, Rho kinase, PI3 kinase, PKC, and PKA. Themethods further comprise blocking survival signaling initiated byligation of integrins by microenvironment proteins. The method alsocomprises use of the inhibitors selected from the group consisting ofLY294002, UO 126, AG82, Y27632, SB203580, PD169316, PD98059, RO318220,and a C3 transferase inhibitor.

Thus, methods of treating primary cancers, metastatic cancers,micrometastases, and hyperproliferative disorders are encompassed by thepresent invention. Combination therapy is also envisioned with otherstandard forms of chemotherapy, radiation therapy and biologicaltherapies and other anti-neoplastic regimens. It is envisioned that thetherapies described in the present invention can be used as adjuncttherapy with other anti-neoplastic treatment modalities.

The roles of various stromal proteins and growth factors that arerelevant to the bone marrow microenvironment in inducing breast cancerdormancy were studied using a panel of breast cancer cell lines.

To test the potential role of FGF-2 in inducing growth arrest of breastcancer cells in the bone marrow microenvironment, the clonogenicpotential of MCF-7, T-47D and MDA-MB-231 breast cancer cells on stromalproteins in the presence of FGF-2 was measured. Clonogenic potential isthe ability of single cells to grow into multi-cell clusters, that is ahallmark of metastatic growth of malignant cells. The presence of FGF-2,but not EGF, significantly blocked clonogenic growth of relativelywell-differentiated MCF-7 and T-47D cells but had no effect on thehighly dedifferentiated aggressive MDA-MB-231 cells. FGF-2 arrestedcells failed to survive on collagen-1 and laminin-1, while they survivedon fibronectin for many days.

To study the molecular basis for the long-term survival of growtharrested cells, a comparison was made between the expression levels ofvarious integrins in breast cancer cells that remained dormant onfibronectin for 3 and 5 days in the presence of FGF-2, to that ofactively growing cells on fibronectin. Microarray analysis showedincreased expression levels of the alpha 5 beta 1 integrin, afibronectin receptor. Western blots demonstrated that FGF-2 induced anincreased expression of both the alpha 5 and beta 1 subunits, whichtogether make up the fibronectin receptor in their naturally pairedstate, in MCF-7 and T-47D cells but had no effect on constitutively veryhigh levels of the alpha 5 subunit in MDA-MB-231 cells. The block ingrowth of FGF-2-treated cells on fibronectin was further accentuated bypre-treatment of the cells with an anti-alpha 5 subunit antibody,strongly suggesting a role for fibronectin in supporting the survival ofdormant breast cancer cells in bone marrow. Blocking peptides thatdisrupt the interaction of fibronectin with its integrin receptor thatdownregulated the expression of the alpha 5 beta 1_integrin alsoreversed the survival effects of fibronectin binding to cells in thepresence of FGF-2. FGF-2 also induced the phosphorylation of the kinaseAkt involved in survival signaling. All trans retinoic acid was able toreverse Akt phosphorylation induced by EGF and reversed FGF-2 inducedincreases in total and Phosphorylated Akt, suggesting an additionalmechanisms of disrupting survival in these cells.

Therapeutic Indications

The administration of kinase or transcription inhibitors or antibodiesor blocking peptides or modified peptides as a pre-treatment tosensitize the dormant or metastatic cells for chemotherapy or radiationtherapy. The inhibitor could be administered in a variety of methodsincluding but not limited to injectable, oral, liquid, tablet orsuppository.

Pharmaceutical Compositions and Methods of Administration

The present invention also provides pharmaceutical compositions used inthe method of the invention. Such compositions comprise atherapeutically effective amount of the agents of the present invention,and a pharmaceutically acceptable carrier. In a particular embodiment,the term “pharmaceutically acceptable” means approved by a regulatoryagency of the Federal or a state government or listed in the U.S.Pharmacopeia or other generally recognized pharmacopeia for use inanimals, and more particularly in humans. The term “carrier” refers to adiluent, adjuvant, excipient, or vehicle with which the therapeutic isadministered. Such pharmaceutical carriers can be sterile liquids, suchas water and oils, including those of petroleum, animal, vegetable orsynthetic origin, such as peanut oil, soybean oil, mineral oil, sesameoil and the like. Water is a preferred carrier when the pharmaceuticalcomposition is administered intravenously. Saline solutions and aqueousdextrose and glycerol solutions can also be employed as liquid carriers,particularly for injectable solutions. Suitable pharmaceuticalexcipients include starch, glucose, lactose, sucrose, gelatin, malt,rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate,talc, sodium chloride, dried skim milk, glycerol, propylene, glycol,water, ethanol and the like. The composition, if desired, can alsocontain minor amounts of wetting or emulsifying agents, or pH bufferingagents.

The therapeutic agent, whether it be a polypeptide, analog or activefragment-containing compositions or small organic molecules, areconventionally administered by various routes including intravenously,intramuscularly, subcutaneously, as by injection of a unit dose, forexample. The term “unit dose” when used in reference to a therapeuticcomposition of the present invention refers to physically discrete unitssuitable as unitary dosage for humans, each unit containing apredetermined quantity of active material calculated to produce thedesired therapeutic effect in association with the required diluent;i.e., carrier, or vehicle.

The compositions are administered in a manner compatible with the dosageformulation, and in a therapeutically effective amount. The quantity tobe administered depends on the subject to be treated, capacity of thesubject's immune system to utilize the active ingredient, and degree ofinhibition or neutralization of binding capacity desired. Preciseamounts of active ingredient required to be administered depend on thejudgment of the practitioner and are peculiar to each individual.Suitable regimes for initial administration and subsequent injectionsare also variable, but are typified by an initial administrationfollowed by repeated doses at intervals by a subsequent injection orother administration.

These compositions can take the form of solutions, suspensions,emulsion, tablets, pills, capsules, powders, sustained-releaseformulations and the like. The composition can be formulated as asuppository, with traditional binders and carriers such astriglycerides. Oral formulation can include standard carriers such aspharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharine, cellulose, magnesium carbonate, etc. Examples ofsuitable pharmaceutical carriers are described in “Remington'sPharmaceutical Sciences” by E. W. Martin. Such compositions will containa therapeutically effective amount of the compound, preferably inpurified form, together with a suitable amount of carrier so as toprovide the form for proper administration to the subject. Theformulation should suit the mode of administration.

The compounds of the invention can be formulated as neutral or saltforms. Pharmaceutically acceptable salts include those formed with freeamino groups such as those derived from hydrochloric, phosphoric,acetic, oxalic, tartaric acids, etc., and those formed with freecarboxyl groups such as those derived from sodium, potassium, ammonium,calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, etc.

Administration of the compositions to the site of injury, the targetcells, tissues, or organs, may be by way of oral administration as apill or capsule or a liquid formulation or suspension. It may beadministered via the transmucosal, sublingual, nasal, rectal ortransdermal route. Parenteral administration may also be via intravenousinjection, or intramuscular, intradermal or subcutaneous. Due to thenature of the diseases or conditions for which the present invention isbeing considered, the route of administration may also involve deliveryvia suppositories. This is especially true in conditions whereby theability of the patient to swallow is compromised.

The plant compositions or extracts may be provided as a liposomeformulation. Liposome delivery has been utilized as a pharmaceuticaldelivery system for other compounds for a variety of applications. See,for example Langer (1990) Science 249:1527-1533; Treat et al. (1989) inLiposomes in the Therapy of Infectious Disease and Cancer,Lopez-Berestein and Fidler (eds.), Liss: New York, pp. 353-365 (1989).Many suitable liposome formulations are known to the skilled artisan,and may be employed for the purposes of the present invention. Forexample, see: U.S. Pat. No. 5,190,762.

In a further aspect, liposomes can cross the blood-brain barrier, whichwould allow for intravenous or oral administration. Many strategies areavailable for crossing the blood-brain barrier, including but notlimited to, increasing the hydrophobic nature of a molecule; introducingthe molecule as a conjugate to a carrier, such as transferrin, targetedto a receptor in the blood-brain barrier; and the like.

Transdermal delivery of the plant compositions or extracts is alsocontemplated. Various and numerous methods are known in the art fortransdermal administration of a drug, e.g., via a transdermal patch. Itcan be readily appreciated that a transdermal route of administrationmay be enhanced by use of a dermal penetration enhancer.

Controlled release oral formulations may be desirable. The plantcomposition or extract may be incorporated into an inert matrix whichpermits release by either diffusion or leaching mechanisms, e.g., gums.Slowly degenerating matrices may also be incorporated into theformulation. Some enteric coatings also have a delayed release effect.Another form of a controlled release of this therapeutic is by a methodbased on the Oros therapeutic system (Alza Corp.), i.e. the drug isenclosed in a semipermeable membrane which allows water to enter andpush drug out through a single small opening due to osmotic effects.

Pulmonary delivery may be used for treatment as well. Contemplated foruse in the practice of this invention are a wide range of mechanicaldevices designed for pulmonary delivery of therapeutic products,including but not limited to nebulizers, metered dose inhalers, andpowder inhalers, all of which are familiar to those skilled in the art.With regard to construction of the delivery device, any form ofaerosolization known in the art, including but not limited to spraybottles, nebulization, atomization or pump aerosolization of a liquidformulation, and aerosolization of a dry powder formulation, can be usedin the practice of the invention.

Ophthalmic and nasal delivery may be used in the method of theinvention. Nasal delivery allows the passage of a pharmaceuticalcomposition of the present invention to the blood stream directly afteradministering the therapeutic product to the nose, without the necessityfor deposition of the product in the lung. Formulations for nasaldelivery include those with dextran or cyclodextrins. For nasaladministration, a useful device is a small, hard bottle to which ametered dose sprayer is attached. In one embodiment, the metered dose isdelivered by drawing the pharmaceutical composition of the presentinvention solution into a chamber of defined volume, which chamber hasan aperture dimensioned to aerosolize and aerosol formulation by forminga spray when a liquid in the chamber is compressed. The chamber iscompressed to administer the pharmaceutical composition of the presentinvention. In a specific embodiment, the chamber is a pistonarrangement. Such devices are commercially available.

The compositions and extracts of the present invention are also suitedfor transmucosal delivery. In particular, the compositions and extractsare particularly suited for sublingual, buccal or rectal delivery ofagents that are sensitive to degradation by proteases present in gastricor other bodily fluids having enhanced enzymatic activity. Moreover,transmucosal delivery systems can be used for agents that have low oralbioavailability. The compositions of the instant invention comprise theplant extract dissolved or dispersed in a carrier that comprises asolvent, an optional hydrogel, and an agent that enhances transportacross the mucosal membrane. The solvent may be a non-toxic alcoholknown in the art as being useful in such formulations of the presentinvention and may include, but not be limited to ethanol, isopropanol,stearyl alcohol, propylene glycol, polyethylene glycol, and othersolvents having similar dissolution characteristics. Other such solventsknown in the art can be found in “The Handbook of PharmaceuticalExcipients”, published by The American Pharmaceutical Association andThe Pharmaceutical Society of Great Britain (1986) and the Handbook ofWater-Soluble Gums and Resins, ed. By R. L. Davidson, McGraw-Hill BookCo., New York, N.Y. (1980).

Any transmucosal preparation suitable for administering the componentsof the present invention or a pharmaceutically acceptable salt thereofcan be used. Particularly, the mixture is any preparation usable inoral, nasal, or rectal cavities that can be formulated usingconventional techniques well known in the art. Preferred preparationsare those usable in oral, nasal or rectal cavities. For example, thepreparation can be a buccal tablet, a sublingual tablet, and the likepreparation that dissolve or disintegrate, delivering drug into themouth of the patient. A spray or drops can be used to deliver the drugto the nasal cavity. A suppository can be used to deliver the mixture tothe rectal mucosa. The preparation may or may not deliver the drug in asustained release fashion.

A specific embodiment for delivery of the components of the presentinvention is a mucoadhesive preparation. A mucoadhesive preparation is apreparation which upon contact with intact mucous membrane adheres tosaid mucous membrane for a sufficient time period to induce the desiredtherapeutic or nutritional effect. The preparation can be a semisolidcomposition as described for example, in WO 96/09829. It can be atablet, a powder, a gel or film comprising a mucoadhesive matrix asdescribed for example, in WO 96/30013. The mixture can be prepared as asyrup that adheres to the mucous membrane.

Suitable mucoadhesives include those well known in the art such aspolyacrylic acids, preferably having the molecular weight between fromabout 450,000 to about 4,000,000, for example, Carbopol™934P; sodiumcarboxymethylcellulose (NaCMC), hydroxypropylmethylcellulose (HPMC), orfor example, Methocel™ K100, and hydroxypropylcellulose.

The delivery of the components of the present invention can also beaccomplished using a bandage, patch, device and any similar device thatcontains the components of the present invention and adheres to amucosal surface. Suitable transmucosal patches are described for examplein WO 93/23011, and in U.S. Pat. No. 5,122,127, both of which are herebyincorporated by reference. The patch is designed to deliver the mixturein proportion to the size of the drug/mucosa interface. Accordingly,delivery rates can be adjusted by altering the size of the contact area.The patch that may be best suited for delivery of the components of thepresent invention may comprise a backing, such backing acting as abarrier for loss of the components of the present invention from thepatch. The backing can be any of the conventional materials used in suchpatches including, but not limited to, polyethylene, ethyl-vinyl acetatecopolymer, polyurethane and the like. In a patch that is made of amatrix that is not itself a mucoadhesive, the matrix containing thecomponents of the present invention can be coupled with a mucoadhesivecomponent (such as a mucoadhesive described above) so that the patch maybe retained on the mucosal surface. Such patches can be prepared bymethods well known to those skilled in the art.

Preparations usable according to the invention can contain otheringredients, such as fillers, lubricants, disintegrants, solubilizingvehicles, flavors, dyes and the like. It may be desirable in someinstances to incorporate a mucous membrane penetration enhancer into thepreparation. Suitable penetration enhancers include anionic surfactants(e.g. sodium lauryl sulphate, sodium dodecyl sulphate), cationicsurfactants (e.g. palmitoyl DL camitine chloride, cetylpyridiniumchloride), nonionic surfactants (e.g. polysorbate 80, polyoxyethylene9-lauryl ether, glyceryl monolaurate, polyoxyalkylenes, polyoxyethylene20 cetyl ether), lipids (e.g. oleic acid), bile salts (e.g. sodiumglycocholate, sodium taurocholate), and related compounds.

The administration of the compositions and extracts of the presentinvention can be alone, or in combination with other compounds effectiveat treating the various medical conditions contemplated by the presentinvention. Also, the compositions and formulations of the presentinvention, may be administered with a variety of analgesics,anesthetics, or anxiolytics to increase patient comfort duringtreatment.

The compositions of the invention described herein may be in the form ofa liquid. The liquid may be delivered as a spray, a paste, a gel, or aliquid drop. The desired consistency is achieved by adding in one ormore hydrogels, substances that absorb water to create materials withvarious viscosities. Hydrogels that are suitable for use are well knownin the art. See, for example, Handbook of Pharmaceutical Excipients,published by The American Pharmaceutical Association and ThePharmaceutical Society of Great Britain (1986) and the Handbook ofWater-Soluble Gums and Resins, ed. By R. L. Davidson, McGraw-Hill BookCo., New York, N.Y. (1980).

Suitable hydrogels for use in the compositions include, but are notlimited to, hydroxypropyl cellulose, hydroxypropyl methyl cellulose,sodium carboxymethyl cellulose and polyacrylic acid. Preferred hydrogelsare cellulose ethers such as hydroxyalkylcellulose. The concentration ofthe hydroxycellulose used in the composition is dependent upon theparticular viscosity grade used and the viscosity desired in the finalproduct. Numerous other hydrogels are known in the art and the skilledartisan could easily ascertain the most appropriate hydrogel suitablefor use in the instant invention.

The mucosal transport enhancing agents useful with the present inventionfacilitate the transport of the agents in the claimed invention acrossthe mucosal membrane and into the blood stream of the patient. Themucosal transport enhancing agents are also known in the art, as notedin U.S. Pat. No. 5,284,657, incorporated herein by reference. Theseagents may be selected from the group of essential or volatile oils, orfrom non-toxic, pharmaceutically acceptable inorganic and organic acids.The essential or volatile oils may include peppermint oil, spearmintoil, menthol, eucalyptus oil, cinnamon oil, ginger oil, fennel oil, dilloil, and the like. The suitable inorganic or organic acids useful forthe instant invention include but are not limited to hydrochloric acid,phosphoric acid, aromatic and aliphatic monocarboxylic or dicarboxylicacids such as acetic acid, citric acid, lactic acid, oleic acid,linoleic acid, palmitic acid, benzoic acid, salicylic acid, and otheracids having similar characteristics. The term “aromatic” acid means anyacid having a 6-membered ring system characteristic of benzene, whereasthe term “aliphatic” acid refers to any acid having a straight chain orbranched chain saturated or unsaturated hydrocarbon backbone.

Other suitable transport enhancers include anionic surfactants (e.g.sodium lauryl sulphate, sodium dodecyl sulphate), cationic surfactants(e.g. palmitoyl DL camitine chloride, cetylpyridinium chloride),nonionic surfactants (e.g. polysorbate 80, polyoxyethylene 9-laurylether, glyceryl monolaurate, polyoxyalkylenes, polyoxyethylene 20 cetylether), lipids (e.g. oleic acid), bile salts (e.g. sodium glycocholate,sodium taurocholate), and related compounds.

When the compositions and extracts of the instant invention are to beadministered to the oral mucosa, the preferred pH should be in the rangeof pH 3 to about pH 7, with any necessary adjustments made usingpharmaceutically acceptable, non-toxic buffer systems generally known inthe art.

For topical delivery, a solution of the agent of the invention in water,buffered aqueous solution or other pharmaceutically-acceptable carrier,or in a hydrogel lotion or cream, comprising an emulsion of an aqueousand hydrophobic phase, at a concentration of between 50 μM and 5 mM, isused. A preferred concentration is about 1 mM. To this may be addedascorbic acid or its salts, or other ingredients, or a combination ofthese, to make a cosmetically-acceptable formulation. Metals should bekept to a minimum. It may be preferably formulated by encapsulation intoa liposome for oral, parenteral, or, preferably, topical administration.

The invention provides methods of treatment comprising administering toa subject a therapeutically effective amount of at least one of theagents described herein. In one embodiment, the compound issubstantially purified (e.g., substantially free from substances thatlimit its effect or produce undesired side-effects). The subject ispreferably an animal, including but not limited to animals such as cows,pigs, horses, chickens, cats, dogs, etc., and is preferably a mammal,and most preferably human. In one specific embodiment, a non-humanmammal is the subject. In another specific embodiment, a human mammal isthe subject.

The amount of the agent of the invention which is optimal in treatingcancers and hyperproliferative disorders can be determined by standardclinical techniques based on the present description. In addition, invitro assays may optionally be employed to help identify optimal dosageranges. The precise dose to be employed in the formulation will alsodepend on the route of administration, and the seriousness of thedisease or disorder, and should be decided according to the judgment ofthe practitioner and each subject's circumstances. However, suitabledosage ranges for intravenous administration are generally about 20-500micrograms of active compound per kilogram body weight. Suitable dosageranges for intranasal administration are generally about 0.01 pg/kg bodyweight to 1 mg/kg body weight. Effective doses may be extrapolated fromdose-response curves derived from in vitro or animal model test systems.

Treatment Group

A subject in whom administration of the agents of the present inventionis an effective therapeutic regiment is preferably a human, but can beany animal. Thus, as can be readily appreciated by one of ordinary skillin the art, the methods and pharmaceutical compositions of the presentinvention are particularly suited to administration to any animal,particularly a mammal, and including, but by no means limited to,domestic animals, such as feline or canine subjects, farm animals, suchas but not limited to bovine, equine, caprine, ovine, and porcinesubjects, wild animals (whether in the wild or in a zoological garden),research animals, such as mice, rats, rabbits, goats, sheep, pigs, dogs,cats, etc., avian species, such as chickens, turkeys, songbirds, etc.,i.e., for veterinary medical use.

Furthermore, the administration of the agent may be given at the time ofor after the identification of a cancer or hyperproliferative disorder,alone, or in combination with other agents known to be beneficial forameliorating the symptoms or decreasing tumor load or enhancing thenumber or activity of immune cells in patients having cancer or ahyperproliferative disorder.

In one embodiment, the subject suitable for treatment by the method ofthe invention is a subject determined to be suffering from cancer orhyperproliferative disorder. This determination may be made clinicallyby methods known to one of skill in the art.

EXAMPLES

The following examples are intended to illustrate the invention notlimit it.

Example 1 FGF-2 Inhibits Single Cell Growth of Well DifferentiatedBreast Cancer Cells

MCF-7 and T-47D cells incubated with FGF-2 have markedly diminishedclonogenic potential in colony assays in tissue culture on laminin-,collagen I- and IV-coated and uncoated plates (FIGS. 2, 3, 4 and 12).The clones that did form in the presence of FGF-2 were arrested in the 8cell stage. FGF-2 had no effect on the growth of the highlyde-differentiated MDA-MB-231 cells. EFG had no effect and served as anegative control in all three cell types.

Example 2 FGF-2 Induces Expression of Cell Integrins Including Integrinα5 and Restricts Growth of Differentiated Single Breast Cancer Cells

Incubation of well differentiated cells with FGF-2 induces theexpression of a variety of cell adhesion molecule genes, including α5,α6, β1 and β3, that contribute to cell death when expressed in anunligated state (FIGS. 5, 6, and Tables 1 and 2). FIG. 6 is a Westernblot demonstrating induction of integrin α5 expression in MCF-7 andT-47D cells growing on either plastic tissue culture dishes orfibronectin-coated dishes. The increase in integrin α5 expression wasassayed for up to five days and remained sustained. No effect isdemonstrated on baseline high levels of integrin α5 in MDA-MB-231 cells.

Example 3 Rescue by Fibronectin

Inhibition of colony formation by FGF-2 can be rescued by incubation ofcells on fibronectin-coated plates (FIGS. 2B, 4, 8, 9, 15 and 16). Theprotection of colonies in MCF-7 cells treated with FGF-2 was sustainedby incubation on fibronectin for up to 15 days (FIG. 8). Fibronectin isa ligand for integrin α5β1 while collagens I and IV are not. These datasuggests an association between unligated integrin α5β1 and inhibitionof growth and rescue of clonogenic potential by providing a specificligand for integrin α5β1.

Example 4 Fibronectin Supports Long-Term Survival of FGF-2 ArrestedCells, Potentially Through a P13K Pathway

Antibody to integrin α5 inhibits the clonogenic potential of MCF-7 cellson fibronectin both with and without FGF-2 treatments (FIG. 7). Antibodyto integrin α3 was used as a negative control. To provide a potentialmechanism for survival signaling by integrin α5 on fibronectin in thepresence of FGF-2, initial experiments were conducted to determine thephosphorylation of Akt by FGF-2 in the presence of fibronectin. FIG. 10demonstrates that FGF-2 induced phosphorylation of Akt in MCF-7 andT-47D. Phosphorylation was sustained for the five days of assay. Highlyde-differentiated MDA-MB-231 cells, however, express constitutivelyhigher levels of integrin α5 and phospho-Akt, implicating thesemolecules in their unlimited growth potential on fibronectin.

Example 5 Disruption of Fibronectin/Integrin α5β1 Interaction canReverse Protection from Cell Death

Our data suggest that stromal proteins in the bone marrowmicroenvironment, such as fibronectin, provide protection of metastaticcancer cells from cell death induced by physiologic factors in the bonemarrow microenvironment and from exogenous toxicity such as chemotherapyor radiation therapy. The ability to disrupt the interaction betweenfibronectin/integrin α5β1 with blocking antibodies to integrin α5 (FIG.7) and β1 (experiments in progress), peptides to the fibronectin bindingsite (FIGS. 8, 9 and 14), antisense phosphorothioated oligonucleotidesto integrins α5 or β1 or downregulation of integrins α5 or β1 in a dosedependent manner, other transcription inhibitors or retinoids, canresult in disruption of the survival signal initiated byfibronectin/integrin α5β1 interaction and thereby become sensitive tochemotherapy and radiation therapy or other biologic therapy-mediatedcell death. This approach may sensitize both well-differentiated cellsthat are non-cycling and dormant in the bone marrow that receivesurvival protection from ligation to fibronectin in the microenvironmentand highly de-differentiated cells that are actively proliferating inthe bone marrow that also receive survival signaling from interactionwith fibronectin through a constitutively upregulated integrin α5.

Example 6 Disruption of the PI3K/Akt Signal Pathway may Disrupt Supportfor Breast Cancer Colony Growth by Fibronectin

FGF-2-induced phosphorylation of Akt may be disrupted in a number ofways by disrupting the interaction of fibronectin with integrin α5β1 bydownregulating the expression of the α5 and β1 subunits, with othertranscription factor inhibitors, retinoids, antisense oligonucleotides,disruption of their interaction with blocking antibodies to the integrinα5 β1 or fibronectin, or kinase inhibitors that inhibit activation ofPI3K or Akt. Examples of Akt inhibition are shown in FIG. 11, whereincubation of MCF-7 cells with ATRA reversed the EGF-mediatedphosphorylation of Akt, as demonstrated on a Western blot, and FIGS. 15and 16 where inhibition of Akt and PI3K, the upstream activation of Aktinhibits survival of dormant clones. This approach may also provide anarray of mechanisms for disruptive survival signaling through the PI3Kpathway to breast cancer cells at metastatic sites initiated byinteraction of integrin α5β1 with fibronectin. Disruption of signalingpathways, kinases and GTPases may disrupt signaling initiated byinteraction of fibronectin with the integrin alpha 5 beta 1 in cancercells that can support survival in these cells. Examples are includedwhich were conducted with inhibitors of Rhp, Rho kinase and MEP/MApkinase, p38, PKC and PKA resulting in the survival of dormant clones onfibronectin (FIGS. 17A and B).

Materials and Methods

Cell Culture

MCF-7, SK-Br-3, MDA-MB-231, PC-3 and LNCaP cells were purchased from theAmerican Type Culture Collection (ATCC), (Rockville, Md.). Cells werecultured in Dulbecco's Modified Eagles Medium (DMEM) (Gibco BRL,Gaithersburg, Md.) with phenol red 15 mg/l, 2 mM glutamine, 10% heatinactivated fetal calf serum (FCS) and penicillin 50 units/ml andstreptomycin 50 micrograms μg/ml (Gemini Bioproducts, Calabasas,Calif.). One to ten thousand cells were incubated on 24 well tissueculture plates that were either commercially coated for tissue culture(uncoated) or coated with 20 □g fibronectin, laminin I, collagen I orcollagen IV, depending on the cell type or experimental conditionsdescribed in the figure legends. Colonies were manually counted at 100×magnification after variable days in culture as described in the figurelegends after removing the media and staining cells with crystal violet.Proliferation kinetics were performed as before¹ using 2% trypan bluecounts on trypsinized cells on the days indicated in the figure intriplicate plates.

Recombinant human FGF-2 and EGF were purchased from R&D Systems,Minneapolis, Minn.). ATRA was purchased from Sigma. Neutralizing mousemonoclonal antibody to integrin α5 or integrin β3 were purchased fromChemicon, Inc. (Temecula, Calif.). Fibronectin-blocking peptide GRGDSPand control peptides were purchased from American Peptide Co., Inc.(Sunnyvale, Calif.).

Western Blots

Cells were harvested and lysates were prepared as described² andanalyzed as before³.

Gene Chip Microarray Analysis

MCF-7 cells were incubated with and without FGF-2 10 ng/ml for 5 days ontissue culture dishes coated with fibronectin 20 μg. Messenger RNA wasprepared using solutions provided in a Nonrad GEArray Q series kit andanalyzed using a Human Extracellular Matrix and Adhesion Protein chipand a Human Pathway Finder chip (Super Array, Bethesda, Md.).

1. A method for disrupting survival signaling from the microenvironmentin cancer cells, wherein said disrupting results in sensitizing cells tochemotherapy, biological therapies or radiation therapy of primarytumors, cancer metastases or micrometastases and hyperproliferativedisorders in a mammal.
 2. The method of claim 1, wherein said methodcomprises blocking the interaction of integrins with the extracellularmatrix proteins of the microenvironment.
 3. The method of claim 2,wherein said integrins are alpha 5 and/or beta 1 integrins and whereinsaid extracellular matrix protein is fibronectin.
 4. The method of claim1, wherein said cancer cell is a breast cancer cell or a prostate cancercell.
 5. The method of claim 2, wherein said method comprisesadministration of an antibody specific for an integrin or a blockingpeptide or modified peptide that disrupts interaction of the integrinwith the extracellular matrix.
 6. The method of claim 5, wherein saidintegrin is an alpha 5 and/or a beta 1 integrin.
 7. A method of claim 1,said method comprising administration of all trans retinoic acid or aretinoic acid derivative.
 8. The method of claim 1, wherein said methodcomprises decreasing expression of cell surface integrins with atranscription inhibitor.
 9. The method of claim 1, wherein said methodcomprises blocking survival signaling initiated by ligation of integrinsby microenvironment proteins.
 10. The method of claim 1, said methodcomprising treatment with an inhibitor of a kinase, said kinase selectedfrom the group consisting of MEP/MAP kinase, p38, RhoA, Rho kinase, PI3kinase, PKC, and PKA.
 11. The method of claim 10, wherein said inhibitoris selected from the group consisting of LY294002, UO 126, AG82, Y27632,SB203580, PD169316, PD98059, RO318220, and a C3 transferase inhibitor.12. A method for treating hyperproliferative disorders in a mammal,comprising administration of an agent capable of blocking the binding ofintegrins with the extracellular matrix.
 13. The method of claim 12,wherein said integrins comprise alpha 5 and/or beta 1 and wherein saidmatrix is fibronectin.
 14. The use of an agent for the preparation of acomposition for treatment of hyperproliferative disorders, said agentcapable of downregulation of the expression of alpha 5 and/or beta 1integrins and their binding to the extracellular matrix.
 15. The use ofkinase or transcription inhibitors as pre-treatment or concurrenttreatment, to sensitize for or potentiate chemotherapy in the treatmentof cancer or hyperproliferative disorders.
 16. The use of claim 15,wherein said cancer is a metastatic cancer.
 17. The use of claim 15,wherein said cancer is breast cancer.
 18. The use of kinase ortranscription inhibitors as pre-treatment or concurrent treatment, tosensitize for or potentiate radiation therapy in the treatment of canceror hyperproliferative disorders
 19. The use of claim 18, wherein saidcancer is a metastatic cancer.
 20. The use of claim 18, wherein saidcancer is breast cancer.
 21. The use of kinase or transcriptioninhibitors to downregulate expression of α5 integrins to treat cancer orhyperproliferative disorders.
 22. The use of claim 21, wherein saidcancer is a metastatic cancer.
 23. The use of claim 21, wherein saidcancer is breast cancer.
 24. The use of kinase or transcriptioninhibitors to downregulate expression of β1 integrins to treat cancer orhyperproliferative disorders.
 25. The use of claim 24, wherein saidcancer is a metastatic cancer.
 26. The use of claim 24, wherein saidcancer is breast cancer.
 27. The use of kinase or transcriptioninhibitors to decrease expression or phosphorylation of Akt in thetreatment of cancer or hyperproliferative disorders.
 28. The use ofclaim 27, wherein said cancer is a metastatic cancer.
 29. The use ofclaim 27, wherein said cancer is breast cancer.
 30. The use of integrinalpha 5 or integrin beta 1 blocking antibodies or blocking peptides ormodified peptides as pre-treatment or concurrent treatment, to sensitizefor or potentiate chemotherapy in the treatment of cancer orhyperproliferative disorders.
 31. The use of claim 30, wherein saidcancer is a metastatic cancer.
 32. The use of claim 30, wherein saidcancer is breast cancer.
 33. The use of integrin alpha 5 or integrinbeta 1 blocking antibodies as pre-treatment or concurrent treatment, tosensitize for or potentiate radiation therapy in the treatment of canceror hyperproliferative disorders
 34. The use of claim 33, wherein saidcancer is a metastatic cancer.
 35. The use of claim 33, wherein saidcancer is breast cancer.
 36. The use of integrin alpha 5 or integrinbeta 1 blocking antibodies or fibronectin blocking peptides or modifiedpeptides as a pre-treatment or concurrent treatment, to sensitize for orpotentiate radiation therapy or chemotherapy in the treatment of cancer.37. The method of claim 36, wherein said cancer is breast cancer. 38.The use of fibronectin binding blocking peptides or modified peptides aspre-treatment or concurrent treatment, to sensitize for or potentiatechemotherapy in the treatment of cancer or hyperproliferative disorders.39. The use of claim 38, wherein said cancer is a metastatic cancer. 40.The use of claim 38, wherein said cancer is breast cancer.
 41. The useof fibronectin binding blocking peptides or modified peptides aspre-treatment or concurrent treatment, to sensitize for or potentiateradiation therapy in the treatment of cancer or hyperproliferativedisorders
 42. The use of claim 41, wherein said cancer is a metastaticcancer.
 43. The use of claim 41, wherein said cancer is breast cancer.44. The use of retinoids and/or retinoid derivatives to decreaseexpression or phosphorylation of Akt in the treatment of cancer orhyperproliferative disorders.
 45. The use of claim 44, wherein saidcancer is a metastatic cancer.
 46. The use of claim 44, wherein saidcancer is breast cancer.
 47. A method of inhibiting cellularproliferation or inducing cell death or cellular differentiation in amammal suffering from a disease or a disorder characterized by cellularproliferation, said method comprising administering a therapeuticallyeffective amount of a kinase or transcription inhibitor prior to, orconcurrent with chemotherapy or radiation therapy.
 48. The method ofclaim 47, wherein said disease or disorder is cancer or ahyperproliferative disorder.
 49. The method of claim 48, wherein saidcancer is breast cancer.
 50. The method of claim 47, wherein said kinaseor transcription inhibitor downregulates expression of alpha 5 integrinsor beta 1 integrins or phosphorylation of Akt to sensitize for orpotentiate chemotherapy or radiation therapy in mammals in need thereof.51. The method of claim 47, wherein said kinase or transcriptioninhibitor is selected from the group consisting of inhibitors of MEP/MAPkinase, p38, RhoA, Rho kinase, PI3 kinase and/or PKC, and PKA.
 52. Themethod of claim 51, wherein said inhibitors are selected from the groupconsisting of LY294002, UO 126, AG82, Y27632, SB203580, PD169316,PD98059, RO318220, and a 3 transferase inhibitor.
 53. A method oftreating cancer or a hyperproliferative disorder in a mammal, the methodcomprising administration of integrin alpha 5 or beta 1 blockingantibodies or fibronectin binding blocking peptides or modifiedpeptides.